siRNA delivery into primary neuronal cells

Functional studies in primary neurons have proved that a novel, highly effective method can now be used to deliver siRNA without a transfection reagent. A straightforward protocol and a growing list of cell-type specific tips will increase success, even in difficult cell types.

	Figure 1. The NTC Red uptake showed almost 100% delivery efficiency.

RNAi technology is a potent tool for functional target validation. In drug discovery for the field of neuroscience, primary neuronal cultures are employed as a cellular model system for the study of neurodegenerative diseases.

The challenge is that primary cultures permit relatively low throughput analysis. Neurons are post-mitotic, highly differentiated cells that require specific growth conditions. Therefore, methods for modification of target expression by over-expression/RNAi are often not efficient enough to obtain robust data employing biochemical assays.

Recently, reporter-based assays were described for the validation of functional effects of modulation of target expression in neurons. However, reporter assays represent an indirect measure of neuronal viability. Moreover, increased transfection-associated toxicity makes accurate evaluation of modulated target expression difficult.

A new technology
Thermo Scientific Dharmacon Accell siRNA, specially modified for delivery to cells without transfection reagents, was investigated for its application in primary rat cortical neurons. Following the Accell siRNA delivery protocol, modified siRNAs are introduced into cells in the optimised Accell delivery medium. Cells are incubated with the delivery mix for at least 48 hours before functional assays. Primary neurons were maintained in the Neurobasal medium™ (Gibco) supplied with B27 (Invitrogen).

A series of experiments were performed to assess the effects of the Accell delivery mix on neuronal viability. Primary cortical neurons at four days in vitro (DIV) were incubated with non-targeting control (NTC) siRNA in the Accell medium alone, complete Neurobasal (NB) medium alone, or in mixes containing different ratios of the two (50:50 or 25:75).

After 48 hours incubation, viability was assessed by visual inspection and MTT assay. The Accell medium alone significantly reduced neuronal viability, yet improved viability was obtained in the 50:50 mix.

siRNA delivery efficiency
Neurons at 4 DIV were incubated for 48 hours with Accell NTC Red siRNA in a 50:50 mix of Accell delivery medium and Neurobasal medium. Cells were fixed and images were acquired using a confocal microscope. Virtually all neurons were positive for NTC Red uptake indicating almost 100% delivery efficiency (Figure 1).

Target knockdown analysis
Neurons at 4 DIV were incubated for 48 hours with the Accell glyceraldehyde 3-phosphate dehydrogenase (GAPD) siRNA in mixes containing different proportions of Accell delivery medium in complete Neurobasal medium (100%, 50%, 25%, 0%).

Quantitative polymerase chain reaction analysis showed significantly decreased expression of the GAPD mRNA compared with NTC. Greater than 90% knockdown was obtained in the mix containing 100% of the Accell delivery medium. However, these conditions do not reflect optimal cellular viability.

For subsequent Accell siRNA delivery experiments, the 50:50 ratio mix represented
a balance between efficiency of knockdown and viability.

Conclusions
Accell siRNA permits delivery to primary cortical neurons with high efficiency. Optimisation of Accell siRNA delivery conditions improves cell viability in primary cortical neurons without compromising target knockdown efficiency.

Accell siRNA delivery technology provides the potential to employ whole culture biochemical assays for functional target validation in primary neurons.